Coxsackievirus-adenovirus Receptor Research Articles

Porcine adenovirus serotype 3 internalization is independent of CAR and α vβ 3 or α vβ 5 integrin

Nonhuman adenoviruses including porcine adenovirus serotype 3 (PAd3) are emerging vectors for gene delivery. PAd3 efficiently transduces human and murine cells in culture, and circumvents preexisting humoral immunity in humans. The coxsackievirus–adenovirus receptor (CAR) serves as a primary receptor and α vβ 3 or α vβ 5 integrin as a secondary receptor for several human adenovirus (HAd) subtypes including HAd5. In this study, we deduced the role of CAR, α vβ 3 or α vβ 5 integrin in PAd3 internalization. Transduction experiments were conducted in human mammary epithelial (MCF-10A) cells using replication-defective PAd-GFP (PAd3 vector expressing green fluorescent protein [GFP]) and HAd-GFP (HAd5 vector expressing GFP). MCF-10A cells were treated with or without anti-human CAR, or anti-α vβ 3 or anti-α vβ 5 integrin antibodies prior to infection with HAd-GFP or PAd-GFP. Significant ( P < 0.05) inhibition in transduction by HAd-GFP was observed in antibody-treated cells as compared to untreated cells, whereas transduction by PAd-GFP remained to similar levels irrespective of the treatment. To study the adenoviral fiber knob-mediated virus interference, MCF-10A cells were treated with or without the recombinant HAd5 or PAd3 knob followed by infection with HAd-GFP or PAd-GFP. Significant ( P < 0.05) inhibition was observed only in transduction of the homologous vector. These results suggested that PAd3 internalization was CAR- as well as α vβ 3 or α vβ 5 integrin-independent and the primary receptor for HAd5 and PAd3 were distinct. CAR- and α vβ 3 or α vβ 5 integrin-independent entry of PAd3 vectors may have implications in targeting cell types that are not efficiently transduced by other adenoviral vectors.

Protein Transduction Domains Fused to Virus Receptors Improve Cellular Virus Uptake and Enhance Oncolysis by Tumor-Specific Replicating Vectors

Expression of cellular receptors determines viral tropism and limits gene delivery by viral vectors. Protein transduction domains (PTDs) have been shown to deliver proteins, antisense oligonucleotides, liposomes, or plasmid DNA into cells. In our study, we investigated the role of several PTD motifs in adenoviral infection. When physiologically expressed, a PTD from human immunodeficiency virus transactivator of transcription (Tat) did not improve adenoviral infection. We therefore fused PTDs to the ectodomain of the coxsackievirus-adenovirus receptor (CAR(ex)) to attach PTDs to adenoviral fiber knobs. CAR(ex)-Tat and CAR(ex)-VP22 allowed efficient adenoviral infection in nonpermissive cells and significantly improved viral uptake rates in permissive cells. Dose-dependent competition of CAR(ex)-PTD-mediated infection using CAR(ex) and inhibition experiments with heparin showed that binding of CAR(ex)-PTD to both adenoviral fiber and cellular glycosaminoglycans is essential for the improvement of infection. CAR(ex)-PTD-treated adenoviruses retained their properties after density gradient ultracentrifugation, indicating stable binding of CAR(ex)-PTD to adenoviral particles. Consequently, the mechanism of CAR(ex)-PTD-mediated infection involves coating of the viral fiber knobs by CAR(ex)-PTD, rather than placement of CAR(ex) domains on cell surfaces. Expression of CAR(ex)-PTDs led to enhanced lysis of permissive and nonpermissive tumor cells by replicating adenoviruses, indicating that CAR(ex)-PTDs are valuable tools to improve the efficacy of oncolytic therapy. Together, our study shows that CAR(ex)-PTDs facilitate gene transfer in nonpermissive cells and improve viral uptake at reduced titers and infection times. The data suggest that PTDs fused to virus binding receptors may be a valuable tool to overcome natural tropism of vectors and could be of great interest for gene therapeutic approaches.

Coxsackie B viruses use multiple receptors to infect human cardiac cells

Viral myocarditis is an inflammatory disease of the heart muscle that can be fatal. The primary viruses that have been linked to myocarditis and dilated cardiomyopathy are the human enteroviruses. The most common viruses associated with this disease are the Coxsackie B viruses and in particular Coxsackievirus B3 and Coxsackievirus B5. Early events in viral infection include attachment of the virus onto cell surface receptors. Even though, CD55 and Coxsackievirus adenovirus receptor protein (CAR) have been identified as receptors for Coxsackievirus B3, the exact mechanisms that Coxsackievirus B3 and B5 use to infect the cardiac muscle are not yet known. In this study, attempts were made to inhibit Coxsackievirus B3 and Coxsackievirus B5 infectivity of cardiac cells by using CAR and CD55 specific antibodies. The results show that these antibodies could not completely inhibit Coxsackievirus B3 and Coxsackievirus B5 binding or infectivity. Furthermore five new proteins have been identified that are used by Coxsackieviruses for binding to cardiac tissue and are distinct from CAR or CD55, leading us to believe that these viruses may use a different set of receptors for infection of cardiac muscle.

Cytokine-mediated downregulation of coxsackievirus-adenovirus receptor in endothelial cells.

Endothelial cells have the ability to change their complement of cell surface proteins in response to inflammatory cytokines. We hypothesized that the expression of the coxsackievirus-adenovirus receptor (CAR), a viral receptor and putative cell-cell adhesion molecule, may be altered during the response of endothelial cells to inflammation. To test this hypothesis, we evaluated CAR protein and mRNA levels in human umbilical vein endothelial cells after they were exposed to tumor necrosis factor alpha, gamma interferon, or a combination of the two cytokines. Flow cytometric and Western blot analyses indicated that cytokine treatment led to a synergistic decrease in CAR protein expression. A Western blot analysis showed that CAR levels decreased to 16% +/- 4% or 1% +/- 4% of the CAR protein levels in untreated cells with either 24 or 48 h of cytokine treatment, respectively. Quantitative reverse transcription-PCR demonstrated that the combination treatment caused CAR mRNA levels to decrease to 21% +/- 12% or 5% +/- 3% of the levels in untreated cells after a 24- or 48-h cytokine treatment, respectively. Reduced CAR expression led to a decrease in adenovirus (Ad) binding of 80% +/- 3% (compared with untreated endothelial cells), with a subsequent decrease in Ad-mediated gene transfer that was dependent on the dose and duration of cytokine treatment but not on the dose of Ad. A similar decrease in CAR protein level and susceptibility to Ad infection was observed in human microvascular endothelial cells, while CAR expression on normal human bronchial epithelial cells or A549 lung epithelial cells was less affected by cytokine treatments. Taken together, the data demonstrate that inflammatory cytokines decrease CAR mRNA and protein expression with a concomitant decrease in Ad binding, reflecting the impact of cell physiology on the function of CAR and the potential effect of inflammation on the ability of Ad to transfer genes to endothelial cells.

Conditionally replication-competent adenoviral vectors with enhanced infectivity for use in gene therapy of melanoma.

To generate vector Ad.Tyr-E1A, which is cytolytic for tyrosinase-positive melanoma cells, we replaced the adenoviral E1A promoter with a human tyrosinase enhancer/promoter. To overcome the low transduction efficiency in populations of melanoma cells that exhibit a low level of the coxsackievirus-adenovirus receptor (CAR), we inserted an RGD-4C peptide into the HI loop of the fiber knob domain of the Ad.Tyr-E1A vector. The resulting vector was named Ad.Tyr-E1A(RGD). As a result of these changes, the transduction efficiency of the RGD-modified vector was increased both in vitro and in vivo. Western blot analysis proved that infection of cells with the Ad.Tyr-E1A(RGD) vector led to expression of the E1A gene selectively in tyrosinase-positive melanoma cell lines, but not in tyrosinase-negative cell lines. The Ad.Tyr-E1A(RGD) vector was as potent in its cytotoxic effect as a tumor nonselective vector (Ad.CMV-E1A) in tyrosinase-positive melanoma cell lines. The Ad.Tyr-E1A(RGD) vector produced a higher vector particle yield in tumor cells than did the Ad.Tyr-E1A vector. Intratumoral injection of the Ad.Tyr-E1A(RGD) vector into xenotransplanted human melanoma tumors led to tumor regression in vivo. The combination of tumor-specific replication and enhanced infectivity generates a more potent CRAD vector for gene therapy of melanoma.

Crystal structure of species D adenovirus fiber knobs and their sialic acid binding sites.

Adenovirus serotype 37 (Ad37) belongs to species D and can cause epidemic keratoconjunctivitis, whereas the closely related Ad19p does not. Primary cell attachment by adenoviruses is mediated through receptor binding of the knob domain of the fiber protein. The knobs of Ad37 and Ad19p differ at only two positions, Lys240Glu and Asn340Asp. We report the high-resolution crystal structures of the Ad37 and Ad19p knobs, both native and in complex with sialic acid, which has been proposed as a receptor for Ad37. Overall, the Ad37 and Ad19p knobs are very similar to previously reported knob structures, especially to that of Ad5, which binds the coxsackievirus-adenovirus receptor (CAR). Ad37 and Ad19p knobs are structurally identical with the exception of the changed side chains and are structurally most similar to CAR-binding knobs (e.g., that of Ad5) rather than non-CAR-binding knobs (e.g., that of Ad3). The two mutations in Ad19p result in a partial loss of the exceptionally high positive surface charge of the Ad37 knob but do not affect sialic acid binding. This site is located on the top of the trimer and binds both alpha(2,3) and alpha(2,6)-linked sialyl-lactose, although only the sialic acid residue makes direct contact. Amino acid alignment suggests that the sialic acid binding site is conserved in several species D serotypes. Our results show that the altered viral tropism and cell binding of Ad19p relative to those of Ad37 are not explained by a different binding ability toward sialyl-lactose.

686. CAR-Expression Is Essential for Tumor Inititation in Lung Cancer Xenografts

Top of pageAbstract Rationale: CAR, the Coxsackievirus Adenovirus Receptor, has primarily been studied in its role as the initial cell surface attachment receptor for Coxsackie and group C-Adenoviruses (Ad). Recent studies indicate that CAR serves to mediate homotypic cell-cell adhesion as a component of the tight junction and/or adherens junction. CAR's role in tumorigenesis has been considered, but no mechanistic studies probing this role have been reported. Experimental Design: Non-small cell lung cancer (NSCLC) cells with exuberant CAR expression (FACS: 99.9% positive w/ MCF 55.2) were stably transfected with control and AS-CAR vectors, and CAR(−)clones isolated (FACS: 1.9% positive w/ MCF 9.6). Cells were phenotypically characterized and tumorigenesis was compared in xenograft models. In separate experiments, CAR was functionally blocked using a monoclonal antibody and a specific ligand (Ad fiber knob), and the impact on tumor formation assessed. Results: Expectedly, CAR (−) cells were refractory to Ad-transduction and demonstrated marked reductions in CAR-mRNA by RT-PCR. Silencing CAR expression in cells that exhibited relatively “high” surface expression of this molecule profoundly inhibited their ability to develop xenografts in host mice (Day 40 tumor volume: CAR (+) cells 574 + 304 mm3, CAR (−) cells 0 mm3; n = 10 scid/scid mice, p<0.001). In survival studies, whereas 5/5 mice sufficed to tumor by day 53 post implantation by CAR (+) cells, all 5/5 mice injected with CAR (−) cells survived (p<0.001). Indeed, 4/5 mice injected with CAR(−)cells were euthanized without tumor on day 120. However, one of 5 mice injected with CAR (−) cells displayed delayed tumor growth beginning at 3 months post-injection, and when this tumor was extirpated and analyzed, CAR-expression had re-emerged. In the second series of experiments, co-injection of NSCLC-cells with high and intermediate surface expression of CAR with a blocking antibody inhibited tumorigenesis in nude mice by these cells (p<0.05). The impact of Ad-fiber knob and siRNA-inhibition of CAR-expression in situ is currently underway. Conclusion: Results to date indicate that CAR subserves a novel biological function in tumor-initiation by lung cancer xenografts. The molecular and cellular mechanisms for this function are being explored.

665. Enhanced Transgene Delivery to Activated Vascular Endothelial Cells In Vitro and In Vivo Using a Novel Strategy To Modify Adenovirus Tropism

The use of dendritic cells (DC) loaded with tumor-associated antigens (TAA) for therapeutic cancer vaccines is particularly attractive because of the central role that DCs play in immunity as antigen presenting cells (APCs). Efficient and specific loading of the DCs with TAA genes is needed for therapeutic cancer vaccines to be successful. Defining ways to target DCs would potentially lesson the amount of vector needed for an effective dose, decrease adverse reactions and/or damage to healthy cells, and lower production costs. DCs are extremely refractory to Ad infection due to the low levels of the primary Ad receptor, coxsackievirus-adenovirus receptor (CAR) on their surface. In this regard, we have successfully augmented the transduction of DCs by re-routing the Ad vector to CD40 that is expressed at relatively high levels on the DC surface. Angiotensin-converting enzyme (ACE), a zinc-metalloprotease with broad substrate specificity, is generally known as an important regulator of blood pressure, fluid, and electrolyte homeostasis. We have also reported a high level of ACE expression in DCs [Danilov, 2003, Exp. Hematol, 31: 1301]. Therefore, we evaluated adenovirus (Ad) targeting of human monocyte-derived DCs via the angiotensin-converting enzyme (ACE, CD143) surface marker. Surface and mRNA expression of ACE were analyzed by flow cytometry and real time PCR, then compared to that of CD40, a well-characterized DC marker. Targeting proteins were developed that have two binding specificities. One part of the targeting protein binds to the fiber of the Ad vector and the other part binds to the ACE expressed on the surface of DCs. Ad vectors were then complexed with the targeting proteins. The resulting ACE-targeted Ad was then used to infect human monocyte-derived DCs. We screened transductional and transcriptional activity of ACE-targeted adenovirus that has luciferase reporter genes under the control of the cytomegarovirus promoter in ΔE1 region (AdCMVLuc) in vitro. ACE surface expression levels were greater than CD40 levels in immature DCs (iDC), however, were less in mature DCs (mDC). The results of luciferase assay demonstrated that ACE-targeting significantly (4-fold) increased the Ad transduction efficiency of iDCs compared to untargeted Ad. This vector approach may thus mediate not only high efficiency gene delivery but also serve a proactive role in DC activation. These results illustrate a novel surface marker on DCs that may be utilized for the therapeutic cancer vaccines.

114. Adenoviral Vectors for Gene Therapy with Improved Entry of Malignant Glioma

An adenovirus serotype 35 (Ad35) vector, which is composed of whole Ad35 belonging to subgroup B, has gained interest as a promising vehicle for gene transfer. This is because Ad35 vectors can circumvent the preexisting immunity against Ad serotype 5 (Ad5). In addition, Ad35 vectors efficiently transduce a wide spectrum of human cells in a coxsackievirus-adenovirus receptor (CAR)-independent manner. This spectrum includes human CD34+ cells and dendritic cells, which are refractory to Ad5 vectors. However, cellular receptors for Ad35 have yet to be identified. Recently, human CD46 (hCD46), a ubiquitously expressed complement regulatory protein, has been identified as a cellular receptor for most subgroup B Ad, including Ad35 (Nat. Med., 2003, 9, S1408-1412, J. Virol. 2003, 77, 9183-9191). We have recently developed a replication-incompetent, recombinant Ad35 vector, and we have reported the properties of Ad35 vector-mediated transduction in vitro and in vivo (Gene Ther. 2003, 10, 1041-1048; Mol. Ther., 2003, 8, 813-821). In the present study, in order to fully evaluate the relationship between the expression levels of hCD46 and Ad35 vector-mediated transduction efficiencies, we examined the role of hCD46 on Ad35 vector-mediated transduction in detail. First, we performed Ad35 vector-mediated transduction into CHO cell lines that stably expressed the hCD46 BC1 or BC2 isoform. After infection with the Ad35 vector, these hCD46-expressing CHO cells showed increases of up to 17-fold in their transduction efficiencies compared with wild-type CHO cells, which are refractory to Ad35 infection. Next, the expression levels of hCD46 in various types of human cells and Ad35 vector-mediated transduction efficiencies were examined. FACS analysis revealed that almost all of the cultured human cells, which mediated efficient transduction with the Ad35 vector, expressed high levels of hCD46, and that the expression level of hCD46 was much lower in SF295 cells (glioblastoma multiforme), which are refractory to the Ad35 vector, than in the other cells. Almost 100% of human bone marrow-CD34+ cells, which are susceptible to the Ad35 vector, also expressed high levels of hCD46. Furthermore, the role of hCD46 was examined using hCD46-transgenic (hCD46-Tg) mice. Normal mouse CD46 expression is limited to the testes, whereas hCD46-Tg mice express hCD46 in a pattern closely mimicking that observed in humans. Transduction of the Ad35 vector into primary hepatocytes and peritoneal macrophages from hCD46-Tg mice resulted in significantly higher transgene expression than that from wild-type mice. Finally, to confirm that hCD46 could act as a cellular receptor in vivo, the Ad35 vector was intraperitoneally administered into hCD46-Tg mice. All the organs examined in the hCD46-Tg mice exhibited higher levels of transgene expression than were exhibited by wild-type mice. These findings demonstrated that the Ad35 vector recognizes hCD46 as a functional receptor in vitro and in vivo. Further in vivo characterization of the Ad35 vector using hCD46-Tg mice would be an important step toward gene therapy applications of the Ad35 vector.

844. Novel Pathways of Cell Entry and Trafficking Utilized by Ad Capsid Proteins in the Absence of the Whole Virus

Cellular infection by Adenovirus serotype 5 (Ad5) is mediated by the viral capsid fiber and penton proteins. Whereas the fiber initiates high affinity binding to cellular Coxsackievirus Adenovirus Receptor (CAR) proteins, the penton binds to secondary integrin receptors, triggering receptor-mediated endocytosis of the virus. We have previously demonstrated that recombinant soluble penton proteins can assemble with plasmid DNA and deliver a reporter gene into cultured cells in the absence of the whole virus.

Binding to decay-accelerating factor is not required for infection of human leukocyte cell lines by enterovirus 70.

Enterovirus 70 (EV70) is one of several human enteroviruses that exhibit a propensity for infecting the central nervous system (CNS). The mechanisms by which neurotropic enteroviruses gain access to and invade the CNS are poorly understood. One possibility is that circulating leukocytes become infected and carry neurotropic enteroviruses to the CNS. We examined the ability of EV70 to infect cell lines derived from lymphoid, myeloid, and monocytic lineages. Most leukocyte cell lines tested bound radiolabeled EV70 and were permissive for EV70 replication, suggesting that EV70, in contrast to other enteroviruses, has an in vitro tropism that includes lymphoid, monocytic, and myeloid cell lines. For some of the cell lines, virus binding and infection correlated with surface expression of decay-accelerating factor (DAF), an attachment protein for EV70 on HeLa cells. However, EV70 also adsorbed to and infected cell lines that expressed little or no DAF. In contrast to what was observed for HeLa cells, neither DAF-specific monoclonal antibodies nor phosphatidylinositol-specific phospholipase C treatment inhibited EV70 binding to permissive leukocyte cell lines, and antibody blockade of DAF had little or no effect on EV70 replication. We also found that neither the human coxsackievirus-adenovirus receptor nor intercellular cell adhesion molecule 1, which mediate the entry of coxsackie B viruses and coxsackievirus A21, respectively, functions as a receptor for EV70. EV70 binding to all cell lines was sensitive to sialidase treatment and to inhibition of O glycosylation by benzyl N-acetyl-alpha-D-galactosaminide. Taken together, these results suggest that a sialylated molecule(s) other than DAF serves as a receptor for EV70 on permissive human leukocyte cell lines.

Basic Study for Next‐Generation Gene Therapy Products

AbstractFor Abstract see ChemInform Abstract in Full Text.

Integrin alpha3beta1 is an alternative cellular receptor for adenovirus serotype 5.

Many adenovirus serotypes enter cells by high-affinity binding to the coxsackievirus-adenovirus receptor (CAR) and integrin-mediated internalization. In the present study, we analyzed the possible receptor function of alpha3beta1 for adenovirus serotype 5 (Ad5). We found that penton base and integrin alpha3beta1 could interact in vitro. In vivo, both Ad5-cell binding and virus-mediated transduction were inhibited in the presence of anti-alpha3 and anti-beta1 function-blocking antibodies, and this occurred in both CAR-positive and CAR-negative cell lines. Peptide library screenings and data from binding experiments with wild-type and mutant penton base proteins suggest that the Arg-Gly-Asp (RGD) in the penton base protein, the best known integrin binding motif, is only part of the binding interface with alpha3beta1, which involved multiple additional contact sites.

General strategy for broadening adenovirus tropism.

In spite of its broad host range, adenovirus type 5 (Ad5) transduces a number of clinically relevant tissues and cell types inefficiently, mostly because of low expression of the coxsackievirus-adenovirus receptor (CAR). To improve gene transfer to such cells, we modified the Ad5 fiber knob to recognize novel receptors. We expressed a functional Ad5 fiber knob domain on the capsid of phage lambda and employed this display system to construct a large collection of ligands in the HI loop of the Ad5 knob. Panning this library on the CAR-negative mouse fibroblast cell line NIH 3T3 resulted in the identification of three clones with increased binding to these cells. Adenoviruses incorporating these ligands in the fiber gene transduced NIH 3T3 cells 2 or 3 orders of magnitude better than the parent vector. The same nonnative tropism was revealed in other cell types, independently of CAR expression. These Ad5 derivatives proved capable of transducing mouse and human primary immature dendritic cells with up to 100-fold increased efficiency.

Basic study for next-generation gene therapy products

Successful gene therapy depends largely on vectors that can efficiently deliver the therapeutic genes into the target tissues and cells. Recombinant adenovirus (Ad) vectors continue to be the preferred vectors for gene therapy because they can easily be grown to high titers and can efficiently transfer genes into both dividing and nondividing cells. However, there are some limitations such as the time-consuming and labor-intensive procedures for vector construction, coxsackievirus-adenovirus receptor (CAR)-dependent gene transfer, immunologic side effects, lack of tissue specificity, lack of regulation of gene expression, etc. In this paper, I review our approach to the development of advanced recombinant Ad vectors. The next generation of Ad vectors have not only become promising vectors for gene therapy but also important tools for gene transfer into mammalian cells.

Heparan sulfates and coxsackievirus-adenovirus receptor: each one mediates coxsackievirus B3 PD infection.

Amino acid exchanges in the virus capsid protein VP1 allow the coxsackievirus B3 variant PD (CVB3 PD) to replicate in decay accelerating factor (DAF)-negative and coxsackievirus-adenovirus receptor (CAR)-negative cells. This suggests that molecules other than DAF and CAR are involved in attachment of this CVB3 variant to cell surfaces. The observation that productive infection associated with cytopathic effect occurred in Chinese hamster ovary (CHO-K1) cells, whereas heparinase-treated CHO-K1 cells, glucosaminoglycan-negative pgsA-745, heparan sulfate (HS)-negative pgsD-677, and pgsE-606 cells with significantly reduced N-sulfate expression resist CVB3 PD infection, indicates a critical role of highly sulfated HS. 2-O-sulfate-lacking pgsF-17 cells represented the cell line with minimum HS modifications susceptible for CVB3 PD. Inhibition of virus replication in CHO-K1 cells by polycationic compounds, pentosan polysulfate, lung heparin, and several intestinal but not kidney HS supported the hypothesis that CVB3 PD uses specific modified HS for entry. In addition, recombinant human hepatocyte growth factor blocked CVB3 PD infection. However, CAR also mediates CVB3 PD infection, because this CVB3 variant replicates in HS-lacking but CAR-bearing Raji cells, infection could be prevented by pretreatment of cells with CAR antibody, and HS-negative pgsD-677 cells transfected with CAR became susceptible for CVB3 PD. These results demonstrate that the amino acid substitutions in the viral capsid protein VP1 enable CVB3 PD to use specific modified HS as an entry receptor in addition to CAR.

Virus-receptor interactions of coxsackie B viruses and their putative influence on cardiotropism.

Specific virus-receptor interactions are important determinants in the pathogenesis of viral infections, influencing the location and initiation of primary infection as well as the viral spread to other target organs in the postviremic phase. Coxsackieviruses of group B (CVB) specifically interact with at least two receptor proteins, the coxsackievirus-adenovirus receptor (CAR) and the decay-accelerating factor (DAF), and cause a broad spectrum of diseases, including acute and chronic myocarditis. In the human heart, CAR is predominantly expressed in intercalated discs, regions of utmost importance for the functional integrity of the heart. Since DAF is abundantly expressed in epithelial and endothelial cells, interaction of cardiotropic CVB with the DAF coreceptor protein, in addition to CAR, could therefore be advantageous to the virus by enhancing viral entry into the heart.

Activation of adenovirus early promoters and lytic phase in differentiated strata of organotypic cultures of human keratinocytes.

Human oncolytic adenoviruses have been used in clinical trials targeting cancers of epithelial origin. To gain a better understanding of the infectious cycle of adenovirus in normal human squamous tissues, we examined the viral infection process in organotypic cultures of primary human keratinocytes. We show that for the infection to occur, wounding of the epithelium is required. In addition, infection appears to initiate at the basal or parabasal cells that express the high-affinity coxsackievirus-adenovirus receptor, CAR, whereas the productive phase takes place in differentiated cells. This is due, at least in part, to the differentiation-dependent activation of the E1A and E2A early promoters and E4 promoters. We also show that adenovirus infection triggers a response mediated by the abnormal accumulation of cyclin E and p21cip1 proteins similar to the one previously observed in human papillomavirus-infected tissues. However, the virus seems to be able to overcome it, at least partially.

Binding of adenoviral fiber knob to the coxsackievirus-adenovirus receptor is crucial for transduction of fetal muscle.

Adenoviral (Ad) infection involves attachment mediated by the Ad fiber protein binding to the coxsackievirus-adenovirus receptor (CAR) of a target cell and internalization facilitated by the interaction of the Ad penton base protein with alpha(v) integrins. To understand the relative importance of the Ad binding and internalization steps for the transduction of fetal skeletal muscle, we used a panel of genetically modified vectors that specifically ablate the fiber-CAR interaction (AdL.F*), the penton base-alpha(v) integrin interaction (AdL.PB*), or both (AdL.PB*F*) to transduce embryonic day 16 (E-16) mouse muscle in vivo and primary E-16 muscle cells in vitro. Quantification of transgene expression and vector genome copies revealed a striking absence of E-16 muscle transduction by AdL.F* and AdL.PB*F*. In contrast, fetal muscle transduction with AdL.PB* was not significantly different than with the unmodified vector. Similar results were observed with in vitro Ad infection studies in primary E-16 muscle cells. From these data we conclude that the fiber-CAR interaction is important for the transduction of fetal muscle by Ad vectors. The high dependence on fiber-CAR binding will impact the development of strategies for Ad vector retargeting to achieve muscle-specific transduction in utero.

Variation in enterovirus receptor genes.

The increased incidence of a enterovirus infections observed in patients with type 1 diabetes preceding the development of the clinical disease could be partially explained by variation in the genes coding for enterovirus receptors. We carried out sequence analysis of the most common enterovirus receptor molecules in 21 diabetic children and 20 healthy adults. DNA was isolated from the leukocytes, and gene regions known to code for virus-recognizing domains in major enterovirus receptors were amplified and sequenced. Heterozygous single-nucleotide polymorphism (SNP), Ala 67 (GCG) --> Thr (ACG), was detected in the poliovirus receptor gene in four individuals in the diabetes group, but not in the control group. However, serological studies could not confirm that this substitution would convey different susceptibility to poliovirus infection. A heterozygous SNP, Lys 29 (AAG) --> Met (ATG), was found in the intracellular adhesion molecule-1 (ICAM-1) (receptor for rhinoviruses and some coxsackie A viruses) in one individual in both groups. A silent SNP in the alpha2 integrin subunit gene (echovirus 1 receptor) was frequently found in both groups, a silent heterozygotic SNP in coxsackievirus-adenovirus receptor (coxsackie B virus receptor) gene was seen in one individual in the diabetes group, whereas no variation was found in the DAF (echovirus receptor) and beta3 integrin subunit sequences (receptor for coxsackievirus A9) studied. In conclusion, both synonymous and nonsynonymous sequence variability of genes coding for enterovirus and rhinovirus receptors was shown to occur, but no pattern directly specific for type 1 diabetes was found. =